P433: ADAPTER HOLIDAYS MAINTAIN IN-VITRO ADCAR-T CELL FUNCTIONALITY AGAINST AML
Authors: D. Nixdorf, M. Sponheimer, D. Berghammer, N. Zieger, L. Rohrbacher, S. Dapa, C. M. Seitz, K. Brandstetter, M. von Bergwelt-Baildon, H. Leonhardt, J. Mittelstaet, A. Kaiser, V. Bücklein, M. Subklewe
Journal: HemaSphere (2022)

Background: The rise of immunotherapies has sparked hope for improving the treatment options of acute myeloid leukemia (AML). However, bispecific antibodies (AB) and CAR-T cells demonstrated only moderate efficacy in clinical trials. The heterogeneous nature of the disease requires more flexible, potent and safer approaches to make T-cell based treatments available for AML patients. We have recently developed a novel, highly modular adapter CAR-T cell (AdCAR) platform capable of targeting multiple antigens via biotin-labeled adapter molecules (AMs) in the context of a specific linker (Seitz et al., 2021). Aims: Utilizing an AdCAR-T cell approach to overcome well described obstacles in AML we evaluated several key parameters of our platform, including cytotoxicity, AM internalization and serial targeting capability. Furthermore, we highlight the possibility of intermittent (int.) AM dosing regimens (AM holidays) to delay AdCAR-T cell dysfunction due to continuous (cont.) stimulation. Methods: The cytotoxic capacity of AdCAR-T against AML cell lines and primary AML (pAML) samples was evaluated in co-culture assays utilizing Fab-based AMs. Specific lysis was assessed by FC after 48-72 h. Internalization kinetics of AMs were assessed via surface staining of AML cells with AMs and secondary staining with anti-Biotin-PE AB. The % surface bound AMs over time at 37 °C vs 4 °C was analyzed. For serial targeting experiments the AMs were either added once, replenished every 3 days, or changed to AMs with different target specificity. To mimic AM holidaysin vitro, cont. vs int. AM-exposed AdCAR-T were co-cultured with OCI-AML-3 (21 d vs 7 d AM exposure, followed by 7 d AM absence). Cytotoxic capacity, T cell proliferation and cytokine secretion were assessed. Results: AdCAR-T cells elicited high cytotoxicity against several AML cell lines utilizing AMs of different specificity (AMs for CD33, CD123, CLL-1; IC50 = 2.1, 0.6, 1.9 ng/ml). Against pAML similar data were obtained in an E:T and AM-concentration dependent manner. We demonstrated feasibility of serial AM usage by performing long-term pAML/AdCAR-T co-cultures. In comparison to one-time or low-dose cont. CD33 AM exposure, which led to an outgrowth of pAML cells after 12 d, increasing AM concentrations and/or switching the target antigen improved AdCAR-T cell efficacy. We further observed receptor-mediated endocytosis of AMs (reduction of MV4-11 surface bound CD33 AM after 6 h = 81%) and postulate that AM internalization is a common form of antigen sink in AM-based T-cell recruiting therapies. Internalization of AMs reduces AM half-life and decreases cytotoxic capacity of AdCAR-T cells. While limited AM levels can be overcome by repetitive dosing, AM internalization potentially also contributes to faster off-switch dynamics. Importantly, we observed that cont. AdCAR-T cell stimulation leads to T cell dysfunction (Fig 1) in in vitro long-term efficacy studies. Strikingly, these effects can be mitigated by implementing int. AM dosing regimens, shown by significantly increased T cell proliferation (fold change d14: 1.9 vs 3.5), cytotoxic capacity (% specific lysis d14: 43 vs 64) and IL-2 production (202 vs 823 pg/ml; all mean+/-SEM). Image:Summary/Conclusion: Taken together, AdCAR-T cells provide flexibility in target antigen choice, safety through short half-life of the AMs and long-term functionality by mitigating AdCAR-T cell dysfunction through AM holidays. Future trials need to consider the high risk of CAR-T cell exhaustion upon cont. stimulation and AdCAR-T cells might pave the way for rational design of more sophisticated treatment schedules.